The action of luteinizing hormone releasing hormone (LHRL) to stimulate luteinizing hormone (LH) secretion by pituitary gonadotrophs is inhibited by testosterone and stimulated by estradiol and progesterone. This steroidal modulation is clearly demonstrable following the administration of physiological concentrations of steroids and LHRH to enzymatically dispersed pituitary cells grown in primary monolayer culture. We hae shown that the actions of estradiol and testosterone depend on both steroid interaction with intracellular receptors and protein synthesis. We plan to use this in vitro model system to expand our studies of the intracellular mechanisms underlying steroid modulation of gonadotroph responsiveness to LHRH. In some studies we will utilize cell populations enriched in gonadotrophs, prepared by velocity sedimentation at unit gravity. Initially we will study progesterone enhancement of LHRH action and examine the role of intracellular receptors and protein synthesis in this effect. Next we will characterize those proteins whose rate of synthesis is influenced by testosterone, estradiol and progesterone by monitoring steroid effects on 35S-methionine incorporation into specific proteins in the gonadotroph using two-dimensional polyacrylamide gel electrophoresis and autoradiography. We will also study LHRH effects on these proteins. Further studies will determine whether synthesis of polyadenylated messenger RNA plays a role in steroid effects on LH secretion and protein synthesis. We will also examine the mechanism of LHRH action to stimulate LH secretion in order to identify steroid-sensitie steps in this process. We will characterize steroid effects on LHRH receptors using a whole cell radioreceptor assay; we will examine steroid influences on the stimulation of LH release by cyclic nucleotides and CA++; and we will study steroid effects on LHRH stimulation of protein kinase activity in the gonadotroph, to determine whether steroid modulation of LHRH action is mediated by alterations in intracellular protein phosphorylation.